Activation of Nuclear Factor-Kappa B Signalling Promotes Cellular Senescence

Oncogene (2011) 30, 2356–2366 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Activation of nuclear factor-kappa B signalling promotes cellular senescence E Rovillain1, L Mansfield1, C Caetano1, M Alvarez-Fernandez2, OL Caballero3, RH Medema2, H Hummerich4 and PS Jat1 1Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK; 2Department of Medical Oncology, University Medical Center, Utrecht, The Netherlands; 3Ludwig Institute for Cancer Research Ltd, New York, NY, USA and 4MRC Prion Unit, UCL Institute of Neurology, London, UK Cellular senescence is a programme of irreversible cell of intrinsic and extrinsic stimuli including progressive cycle arrest that normal cells undergo in response to shortening of telomeres or changes in telomeric struc- progressive shortening of telomeres, changes in telomeric ture or other forms of genotoxic stress such as oncogene structure, oncogene activation or oxidative stress. The activation, DNA damage or oxidative stress resulting in underlying signalling pathways, of major clinicopatholo- a DNA damage response and growth arrest via gical relevance, are unknown. We combined genome-wide activation of the p53 tumour suppressor (Ben-Porath expression profiling with genetic complementation to and Weinberg, 2005; Campisi and d’Adda di Fagagna, identify genes that are differentially expressed when 2007; Adams, 2009). Activation of p53 induces p21CIP1, conditionally immortalised human fibroblasts undergo which prevents cyclin/cdk complexes from phosphor- senescence upon activation of the p16-pRB and p53-p21 ylating the RB family of proteins, thereby activating the tumour suppressor pathways. This identified 816 up and pRB tumour suppressor pathway (Ben-Porath and 961 downregulated genes whose expression was reversed Weinberg, 2005; Campisi and d’Adda di Fagagna, when senescence was bypassed. Overlay of this data set 2007; Adams, 2009). Under other circumstances, the with the meta-signatures of genes upregulated in cancer pRB pathway can be activated independently of p53, showed that nearly 50% of them were downregulated upon through upregulation of p16INK4A, an inhibitor senescence showing that even though overcoming senes- of the cyclinD/cdk4,6 kinases that also phosphorylate cence may only be one of the events required for malignant the RB family of proteins (Ben-Porath and Weinberg, transformation, nearly half of the genes upregulated in 2005; Campisi and d’Adda di Fagagna, 2007; Adams, cancer are related to it. Moreover 65 of the up and 26 of 2009). the downregulated genes are known downstream targets of Senescence can compromise tissue repair and regen- nuclear factor (NF)-jB suggesting that senescence was eration and contribute to tissue and organismal ageing associated with activation of the NF-jB pathway. Direct due to depletion of stem/progenitor cell compartments. perturbation of this pathway bypasses growth arrest It could also lead to removal of defective and potentially indicating that activation of NF-jB signalling has a cancerous cells from the proliferating pool thereby causal role in promoting senescence. limiting tumour development (Campisi and d’Adda di Oncogene (2011) 30, 2356–2366; doi:10.1038/onc.2010.611; Fagagna, 2007). Acquisition of a limitless replicative published online 17 January 2011 potential has been proposed to be one of the key events required for malignant transformation (Hanahan and Keywords: cellular senescence; conditional immortalisa- Weinberg, 2000). The underlying mechanisms that tion; senescence bypass; differential expression, NF-kB control cellular senescence, the signal transduction signalling pathways involved and how the diverse signals that result in senescence are all integrated, remain poorly defined. Introduction The main stumbling block in studying senescence has been the absence of suitable model systems because it is Cellular senescence is a programme of irreversible cell stochastic and occurs asynchronously in heterogeneous cycle arrest that normal cells undergo after a finite cell populations that are typically used for serial sub- number of divisions, the Hayflick limit (Hayflick and cultivation. Studies with human cells are further Moorhead, 1961). It is triggered in response to a variety complicated by the genetic, epigenetic and proliferative variation that can exist between different donors, as well Correspondence: Professor PS Jat, Department of Neurodegenerative as phenotypic differences between cells within the Disease, UCL Institute of Neurology, Queen Square, London WC1N cultures. To simplify this process many investigators 3BG, UK. study oncogene-induced senescence due to expression of E-mail: [email protected] V12 E600 Received 27 July 2010; revised 10 November 2010; accepted 4 December activated oncogenes such as RAS , RAF or BRAF 2010; published online 17 January 2011 or stress or irradiation induced senescence where it NF-jB signalling promotes senescence E Rovillain et al 2357 occurs prematurely and can be induced acutely in a Results variety of cell types (Collado et al., 2007). We have taken a different approach by making use of Senescence is predominantly induced by the p53-p21 the finding that reconstitution of telomerase activity by pathway the catalytic subunit of human telomerase (hTERT) To define the relative contributions of the p16-pRB and alone was incapable of immortalising all human somatic p53-p21 pathways towards senescence, each of these cells, but inactivation of the p16-pRB and p53-p21 pathways was abrogated by ectopic expression or pathways were required in addition (Counter et al., RNAi. To facilitate efficient retroviral infection, 1998; Kiyono et al., 1998). The ability of SV40 large T HMF3A cells were transduced with the full length (LT) antigen to inactivate the p16-pRB and p53-p21 murine ecotropic retroviral receptor (receptor expressing pathways enabled the use of a thermolabile mutant cells are designated as EcoR) and 24 single cell clones (U19tsA58) of LT antigen, in conjunction with hTERT, analysed for temperature dependent growth and infect- to develop conditionally immortalised human mammary ibility with ecotropic retroviruses. Although all clones (HMF3A) fibroblasts, that remain stringently tempera- exhibited temperature dependent growth, clone 3 ture sensitive and show no sign of transformation in (CL3EcoR) cells most closely mirrored the parental cells 4300 population doublings (O’Hare et al., 2001). These in their temperature dependent growth characteristics cells are immortal if grown at 34 1C but upon inactiva- (Figures 1a–d). They undergo an irreversible growth tion of the thermolabile LT antigen they undergo an arrest upon shift to 38 1C and exhibit the same changes irreversible growth arrest within 7 days, induce senes- in morphology as the HMF3A cells and express cence-associated b-galactosidase and have morphologi- senescence-associated b-galactosidase (Figure 1e). cal features and express genes in common with senescent Complementation analysis indicated that the tem- cells (Hardy et al., 2005). perature dependent growth defect was readily overcome Previously we used complementary DNA microarrays with wild type SV40 LT antigen. Different numbers of to identify genes that were differentially expressed when stably transduced cells were seeded and cultured at 38 1C these conditionally immortal HMF3A fibroblasts under- for 3 weeks; densely growing clones were observed after went irreversible growth arrest upon activation of the plating 1000 stably transduced HMF3AEcoR cells p16-pRB and p53-p21 pathways (Hardy et al., 2005). (Figure 1f). Growth arrest was also very efficiently Although many of the identified changes had previously overcome upon inactivation of the p53-p21 pathway been found to occur upon replicative senescence with p53GSE, that inactivates p53 (Ossovskaya et al., (Shelton et al., 1999), we identified a number of genes 1996) or short-hairpin (sh) RNAs that target p53 (Berns not previously implicated in senescence and in silico et al., 2004) or p21CIP1 (Mansfield, 2006; Figures 1f promoter analysis coupled with electrophoretic mobility and g). This abrogation was very efficient as clones shift assays suggested that nuclear factor-kB (NF-kB) of densely growing cells were detected after plating and C/EBP transcription factors may be activated upon o3000 cells. In contrast, growth arrest was much less senescence (Hardy et al., 2005). Subsequently NF-kB efficiently overcome upon inactivation of the p16-pRB activity was shown to be continually required to enforce pathway (Figure 1f); growing cells were only obtained many features of ageing in a tissue specific manner after plating 30–50 000 cells, stably transduced with (Adler et al., 2007) and be constitutively activated adenovirus 5 E1A 12s, human papilloma virus (HPV) 16 upon ageing (Kriete et al., 2008; Salminen et al., 2008; E7 or the dominant negative E2F-DB protein (Rowland Kriete and Mayo, 2009). Association of senescence et al., 2002). Silencing of p21 expression by p53shRNA, with secretion of senescence-associated-secretory- p53GSE and p21shRNA is shown in Supplementary phenotype proteins further suggested a role for NF-kB Figure S1; expression of E1A and E7 are shown in activation in inducing and reinforcing senescence Supplementary Figure. S2. (Kuilman and Peeper, 2009). In contrast, others have Growth arrest of CL3EcoR cells was also readily obtained contradictory results (Dimri and Campisi, bypassed by SV40 LT antigen, p53GSE, p53shRNA or 1994;

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